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Silibinin Strongly Synergizes Human Prostate Carcinoma DU145 Cells to Doxorubicin-induced Growth Inhibition, G₂-M Arrest, and Apoptosis¹

Department of Pharmaceutical Sciences, School of Pharmacy [A. K. T., R. P. S., C. A., R. A.], Division of Medical Oncology, Department of Medicine [D. C. F. C.], and University of Colorado Cancer Center [D. C. F. C., R. A.], University of Colorado Health Sciences Center, Denver, Colorado 80262

Purpose: We recently demonstrated the strong anticancer efficacy of silibinin,an active constituent of a widely consumed dietary supplement milk thistle extract, against human prostate cancer cells in culture and nude mice xenografts. We also observed that pharmacologically achievable concentrations of silibinin in animal studies were in the range of 25–100 µM, depending on the dose regimen, which did not show any apparent toxicity to the animals. In this study, we assessed whether silibinin synergizes the therapeutic potential of the chemotherapeutic drug doxorubicin against prostate cancer, the effectiveness of which is limited because of high systemic toxicity.

Experimental Design: Prostate cancer cells were treated with silibinin and doxorubicin, either alone or in combination, and cell growth was determined by manual cell counting. Cell cycle progression was assessed by saponin/propidium iodide staining and fluorescence-activated cell sorter analysis. Protein levels of cell cycle regulators were determined by Western blotting, and cdc2/p34 kinase activity was analyzed by in-beads kinase assay. Apoptosis was quantified by annexin V/propidium iodide staining and fluorescence-activated cell sorter analysis.

Results: Silibinin strongly synergized the growth-inhibitory effect of doxorubicin in prostate carcinoma DU145 cells (combination index, 0.235–0.587), which was associated with a strong G₂-M arrest in cell cycle progression, showing 88% cells in G₂-M phase by this combination compared with 19 and 41% of cells in silibinin and doxorubicin treatment alone, respectively. The underlying mechanism of G₂-M arrest showed a strong inhibitory effect of combination on cdc25C, cdc2/p34, and cyclin B1 protein expression and cdc2/p34 kinase activity. More importantly, this combination caused 41% apoptotic cell death compared with 15% by either agent alone. Silibinin and doxorubicin alone as well as in combination were also effective in inhibiting the growth of androgen-dependent prostate carcinoma LNCaP cells.

Conclusion: These findings suggest a need for in vivo studies with this combination in preclinical prostate cancer models. Positive outcomes might be relevant for a clinical application in prostate cancer patients.

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